Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA-MurG interaction.

Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to "resensitize" enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure-function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA-MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.

The tongue of the red-eared slider (Trachemys scripta elegans): morphological characterization through gross, light, scanning electron, and immunofluorescence microscopic examination.

The red-eared slider (Trachemys scripta elegans) is renowned for its remarkable adaptations, yet much of its complex biology remains unknown. In this pioneering study, we utilized a combination of gross anatomy, scanning electron microscopy (SEM), light microscopy, and immunofluorescence techniques to examine the tongue's omnivorous adaptation in this species. This research bridges a critical knowledge gap, enhancing our understanding of this intriguing reptile. Gross examination revealed a unique arrowhead-shaped tongue with a median lingual fissure and puzzle-piece-shaped tongue papillae. SEM unveiled rectangular filiform, conical, and fungiform papillae, with taste pores predominantly on the dorsal surface and mucous cells on the lateral surface of the papillae. Histologically, the tongue's apex featured short rectangular filiform and fungiform papillae, while the body exhibited varying filiform shapes and multiple taste buds on fungiform papillae. The tongue's root contained lymphatic tissue with numerous lymphocytes surrounding the central crypt, alongside lingual skeletal musculature, blood and lymph vessels, and Raffin corpuscles in the submucosa. The lingual striated muscle bundles had different orientations, and the lingual hyaline cartilage displayed a bluish coloration of the ground substance, along with a characteristic isogenous group of chondrocytes. Our research represents the first comprehensive application of immunofluorescence techniques to investigate the cellular intricacies of the red-eared slider's tongue by employing seven distinct antibodies, revealing a wide array of compelling and significant findings. Vimentin revealed the presence of taste bud cells, while synaptophysin provided insights into taste bud and nerve bundle characteristics. CD34 and PDGFRα illuminated lingual stromal cells, and SOX9 and PDGFRα shed light on chondrocytes within the tongue's cartilage. CD20 mapped B-cell lymphocyte distribution in the lingual tonsil, while alpha smooth actin (α-SMA) exposed the intricate myofibroblast and smooth muscle network surrounding the lingual blood vessels and salivary glands. In conclusion, our comprehensive study advances our knowledge of the red-eared slider's tongue anatomy and physiology, addressing a significant research gap. These findings not only contribute to the field of turtle biology but also deepen our appreciation for the species' remarkable adaptations in their specific ecological niches.

The ASCC2 CUE domain in the ALKBH3-ASCC DNA repair complex recognizes adjacent ubiquitins in K63-linked polyubiquitin.

Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3-Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain. The basis for polyubiquitin-binding specificity was unclear, because CUE domains in other proteins typically bind a single ubiquitin and do not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. The ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, whereas the contacts with the proximal ubiquitin are unique to ASCC2. Residues in the N-terminal portion of the ASCC2 α1 helix contribute to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response. Our study reveals the versatility of CUE domains in ubiquitin recognition.

Liquid-Liquid Phase Separation of the DEAD-Box Cyanobacterial RNA Helicase Redox (CrhR) into Dynamic Membraneless Organelles in Synechocystis sp. Strain PCC 6803.

Compartmentalization of macromolecules into discrete non-lipid-bound bodies by liquid-liquid phase separation (LLPS) is a well-characterized regulatory mechanism frequently associated with the cellular stress response in eukaryotes. In contrast, the formation and importance of similar complexes is just becoming evident in bacteria. Here, we identify LLPS as the mechanism by which the DEAD-box RNA helicase, cyanobacterial RNA helicase redox (CrhR), compartmentalizes into dynamic membraneless organelles in a temporal and spatial manner in response to abiotic stress in the cyanobacterium Synechocystis sp. strain PCC 6803. Stress conditions induced CrhR to form a single crescent localized exterior to the thylakoid membrane, indicating that this region is a crucial domain in the cyanobacterial stress response. These crescents rapidly dissipate upon alleviation of the stress conditions. Furthermore, CrhR aggregation was mediated by LLPS in an RNA-dependent reaction. We propose that dynamic CrhR condensation performs crucial roles in RNA metabolism, enabling rapid adaptation of the photosynthetic apparatus to environmental stresses. These results expand our understanding of the role that functional compartmentalization of RNA helicases and thus RNA processing in membraneless organelles by LLPS-mediated protein condensation performs in the bacterial response to environmental stress. IMPORTANCE Oxygen-evolving photosynthetic cyanobacteria evolved ~3 billion years ago, performing fundamental roles in the biogeochemical evolution of the early Earth and continue to perform fundamental roles in nutrient cycling and primary productivity today. The phylum consists of diverse species that flourish in heterogeneous environments. A prime driver for survival is the ability to alter photosynthetic performance in response to the shifting environmental conditions these organisms continuously encounter. This study demonstrated that diverse abiotic stresses elicit dramatic changes in localization and structural organization of the RNA helicase CrhR associated with the photosynthetic thylakoid membrane. These dynamic changes, mediated by a liquid-liquid phase separation (LLPS)-mediated mechanism, reveal a novel mechanism by which cyanobacteria can compartmentalize the activity of ribonucleoprotein complexes in membraneless organelles. The results have significant consequences for understanding bacterial adaptation and survival in response to changing environmental conditions.

Peptidic Inhibitors and a Fluorescent Probe for the Selective Inhibition and Labelling of Factor XIIIa Transglutaminase.

Factor XIIIa (FXIIIa) is a transglutaminase of major therapeutic interest for the development of anticoagulants due to its essential role in the blood coagulation cascade. While numerous FXIIIa inhibitors have been reported, they failed to reach clinical evaluation due to their lack of metabolic stability and low selectivity over transglutaminase 2 (TG2). Furthermore, the chemical tools available for the study of FXIIIa activity and localization are extremely limited. To combat these shortcomings, we designed, synthesised, and evaluated a library of 21 novel FXIIIa inhibitors. Electrophilic warheads, linker lengths, and hydrophobic units were varied on small molecule and peptidic scaffolds to optimize isozyme selectivity and potency. A previously reported FXIIIa inhibitor was then adapted for the design of a probe bearing a rhodamine B moiety, producing the innovative KM93 as the first known fluorescent probe designed to selectively label active FXIIIa with high efficiency (kinact/KI = 127,300 M-1 min-1) and 6.5-fold selectivity over TG2. The probe KM93 facilitated fluorescent microscopy studies within bone marrow macrophages, labelling FXIIIa with high efficiency and selectivity in cell culture. The structure-activity trends with these novel inhibitors and probes will help in the future study of the activity, inhibition, and localization of FXIIIa.

Quantum Dot-Antibody Conjugates for Immunofluorescence Studies of Biomolecules and Subcellular Structures.

Quantum dots, or nanoscale semiconductors, are one of the most important materials for various research and development purposes. Due to their advantageous photoluminescence and electronic properties, namely, their unique photostability, high brightness, narrow emission spectra from visible to near-infrared wavelengths, convey them significant advantages over widely used fluorochromes, including organic dyes, fluorescent probes. Quantum dots are a unique instrument for a wide range of immunoassays with antibodies. The paper provides an overview of the developed and already applied methods of quantum dot surface modification, quantum dots conjugation to different antibodies (non-covalent, direct covalent linkage or with the use of special adapter molecules), as well as practical examples of recent quantum dot-antibody applications in the immunofluorescence microscopy for cell and cell structure imaging, fluorescent assays for biomolecules detection and in diagnostics of various diseases. The review presents advantages of quantum dot-antibody conjugation technology over the existing methods of immunofluorescence studies and a forward look into its potential prospects in biological and biomedical research.

Heteromannans are the predominant hemicelluloses in the gametophytic stem of the umbrella moss Hypnodendron menziesii and occur in the walls of all cell types.

MAIN CONCLUSION: Heteromannans are the predominant hemicelluloses in the gametophytic stem of the moss Hypnodendron menziesii and occur in the walls of all cell types Little is known about the cell-wall polysaccharides of mosses. Monosaccharide analysis of cell walls isolated from the stem of the umbrella moss Hypnodendron menziesii was consistent with heteromannans, probably galactoglucomannans, being the predominant hemicellulosic polysaccharides in the walls. Immunofluorescence and immunogold microscopy with the monoclonal antibody LM21, specific for heteromannans, showed that these polysaccharides were present in the walls of all stem cell types. These cell types, except the hydroids, have secondary walls. Experiments in which sections were pre-treated with 0.1 M sodium carbonate and with the enzyme pectate lyase indicated that the heteromannans have O-acetyl groups that limit LM21 binding and the cell walls contain pectic homogalacturonan that masks detection of heteromannans using LM21. Therefore, to fully detect heteromannans in the cell walls, it was essential to use these pre-treatments to remove the O-acetyl groups from the heteromannans and pectic homogalacturonan from the cell walls. Fluorescence microscopy experiments with a second monoclonal antibody, LM22, also specific for heteromannans, showed similar results, but the binding was considerably weaker than with LM21, possibly as a result of subtle structural differences in the epitopes of the two antibodies. Although heteromannans occur abundantly in the cell walls of many species in basal lineages of tracheophytes, prior to the present study, research on the distribution of these polysaccharides in the walls of different cell types in mosses was confined to the model species Physcomitrium patens.

New-generation diagnostics in inflammatory skin diseases: Immunofluorescence and histopathological assessment using ex vivo confocal laser scanning microscopy in cutaneous lupus erythematosus.

Ex vivo confocal laser scanning microscopy (CLSM) offers real-time examination of excised tissue in reflectance, fluorescence and digital haematoxylin-eosin (H&E)-like staining modes enabling application of fluorescent-labelled antibodies. We aimed to assess the diagnostic performance of ex vivo CLSM in identifying histopathological features and lupus band test in cutaneous lupus erythematosus (CLE) with comparison to conventional histopathology and direct immunofluorescence (DIF). A total of 72 sections of 18 CLE patients were stained with acridine orange (AO), anti-IgG, anti-IgM and anti-IgA; 21 control samples were stained with AO. Subsequently, ex vivo CLSM examination of all samples was performed in reflectance, fluorescence and digital H&E-like staining modes. Superficial and deep perivascular inflammatory infiltration (94.4%), interface dermatitis (88.9%), spongiosis (83.3%) and vacuolar degeneration (77.7%) were the most common features detected with ex vivo CLSM. Kappa test revealed a level of agreement ranging within "perfect" to "good" between ex vivo CLSM and conventional histopathology. ROC analysis showed that the combination of perivascular infiltration, interface dermatitis and spongiosis detected by ex vivo CLSM has the potential to distinguish between CLE and controls. Basement membrane immunoreactivity with IgG, IgM and IgA was identified in 88.8% (n = 15), 55.5% (n = 10) and 55.5% (n = 10) of the CLE samples using ex vivo CLSM, respectively, whereas DIF showed IgG, IgM and IgA positivity in 94.4% (n = 17), 100% (n = 18) and 88.9% (n = 16) of patients, respectively. In conclusion, ex vivo CLSM enables simultaneous histopathological and immunofluorescence examination in CLE showing a high agreement with conventional histopathology, albeit with a lower performance than conventional DIF.

Diagnostic Value and Practicability of Serration Pattern Analysis by Direct Immunofluorescence Microscopy in Pemphigoid Diseases.

In pemphigoid diseases, direct immunofluorescence can be used to differentiate 2 patterns of antibody deposition at the dermal-epidermal junction; u- and n-serrated pattern. The u-serrated pattern is found in epidermolysis bullosa acquisita, and n-serrated pattern in all other pemphigoid diseases. To determine the detection frequency of these serrated patterns and the optimal thickness of biopsy cryosections, 2 patient cohorts obtained form our routine autoimmune laboratory were analysed; a retrospective cohort (n = 226) and a prospective cohort (n = 156). [AQ1] In 76% (291/382) of biopsies, a pattern was recog-nized, of which 96% (278/291) and 4% (13/291) had an n- or u-serrated pattern, respectively. A u-serrated pattern was seen in all epidermolysis bullosa acquisita biopsies confirmed by serology. No antibodies against type VII collagen were detected in any of the sera from biopsies with n-serrated pattern. No differences between the detection frequencies of serrated pattern were seen with respect to age, sex, biopsy site, or section thickness, while the detection frequency was higher in patients with serum anti-BP180 reactivity compared with those without. In conclusion, serrated pattern analysis using direct immunofluorescence has a high detection frequency and specificity for epidermolysis bullosa acquisita and will further facilitate the diagnosis of latter disorder.

Autolysis of Chlorella variabilis in Starving Paramecium bursaria Help the Host Cell Survive Against Starvation Stress.

The endosymbiosis between Paramecium bursaria and Chlorella spp. is mutualistic. Symbiotic algae localize beneath the host Paramecium cell cortex compete for their attachment sites with preexisting organelle trichocysts. To examine the relationship between P. bursaria trichocysts and their symbiotic algae, algae-bearing or alga-free P. bursaria were starved for several days and the changes in the number of Chlorella sp. and presence or absence of trichocysts were evaluated. We conducted an indirect immunofluorescence microscopy with an anti-trichocyst monoclonal antibody against P. bursaria cells. Indirect immunofluorescence microscopy demonstrated that under starvation and darkness conditions, the immunofluorescence of trichocysts in alga-free P. bursaria decreased much faster than that in the normal algae-bearing P. bursaria. In the latter case, our observations proposed the possibility that the nutrition obtained from symbiotic algal digestion may promote trichocysts synthesis. This algal digestion mechanism may permit host P. bursaria cells to survive for a longer time under starvation condition. To the best of our knowledge, this may be a new benefit that host P. bursaria gain from harboring symbiotic algae.

Synthesis and Characterization of Bis-Triazolyl-Pyridine Derivatives as Noncanonical DNA-Interacting Compounds.

Besides the well-known double-helical conformation, DNA is capable of folding into various noncanonical arrangements, such as G-quadruplexes (G4s) and i-motifs (iMs), whose occurrence in gene promoters, replication origins, and telomeres highlights the breadth of biological processes that they might regulate. Particularly, previous studies have reported that G4 and iM structures may play different roles in controlling gene transcription. Anyway, molecular tools able to simultaneously stabilize/destabilize those structures are still needed to shed light on what happens at the biological level. Herein, a multicomponent reaction and a click chemistry functionalization were combined to generate a set of 31 bis-triazolyl-pyridine derivatives which were initially screened by circular dichroism for their ability to interact with different G4 and/or iM DNAs and to affect the thermal stability of these structures. All the compounds were then clustered through multivariate data analysis, based on such capability. The most promising compounds were subjected to a further biophysical and biological characterization, leading to the identification of two molecules simultaneously able to stabilize G4s and destabilize iMs, both in vitro and in living cells.

SPEF2- and HYDIN-Mutant Cilia Lack the Central Pair-associated Protein SPEF2, Aiding Primary Ciliary Dyskinesia Diagnostics.

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous chronic destructive airway disease. PCD is traditionally diagnosed by nasal nitric oxide measurement, analysis of ciliary beating, transmission electron microscopy (TEM), and/or genetic testing. In most genetic PCD variants, laterality defects can occur. However, it is difficult to establish a diagnosis in individuals with PCD and central pair (CP) defects, and alternative strategies are required because of very subtle ciliary beating abnormalities, a normal ciliary ultrastructure, and normal situs composition. Mutations in HYDIN are known to cause CP defects, but the genetic analysis of HYDIN variants is confounded by the pseudogene HYDIN2, which is almost identical in terms of intron/exon structure. We have previously shown that several types of PCD can be diagnosed via immunofluorescence (IF) microscopy analyses. Here, using IF microscopy, we demonstrated that in individuals with PCD and CP defects, the CP-associated protein SPEF2 is absent in HYDIN-mutant cells, revealing its dependence on functional HYDIN. Next, we performed IF analyses of SPEF2 in respiratory cells from 189 individuals with suspected PCD and situs solitus. Forty-one of the 189 individuals had undetectable SPEF2 and were subjected to a genetic analysis, which revealed one novel loss-of-function mutation in SPEF2 and three reported and 13 novel HYDIN mutations in 15 individuals. The remaining 25 individuals are good candidates for new, as-yet uncharacterized PCD variants that affect the CP apparatus. SPEF2 mutations have been associated with male infertility but have not previously been identified to cause PCD. We identified a mutation of SPEF2 that is causative for PCD with a CP defect. We conclude that SPEF2 IF analyses can facilitate the detection of CP defects and evaluation of the pathogenicity of HYDIN variants, thus aiding the molecular diagnosis of CP defects.

Hyperkeratotic hand eczema: Eczema or not?

BACKGROUND: Hyperkeratotic hand eczema (HHE) is a typical clinical hand eczema subtype with a largely unknown pathophysiology. OBJECTIVE: To investigate histopathology, expression of keratins (K), epidermal barrier proteins, and adhesion molecules in HHE. METHODS: Palmar skin biopsies (lesional and perilesional) were obtained from seven HHE patients and two healthy controls. Moreover, 135 candidate genes associated with palmoplantar keratoderma were screened for mutations. RESULTS: Immunofluorescence staining showed a significant reduction of K9 and K14 in lesional skin. Upregulation was found for K5, K6, K16, and K17 in lesional skin compared with perilesional and healthy palmar skin. Further, upregulation of involucrin and alternating loricrin staining, both in an extracellular staining pattern, was found. Filaggrin expression was similar in lesional, perilesional, and control skin. No monogenetic mutations were found. CONCLUSION: Currently, the phenotype of HHE is included in the hand eczema classification system; however, it can be argued whether this is justified. The evident expression of filaggrin and involucrin in lesional skin does not support a pathogenesis of atopic eczema. The upregulation of K6, K16, and K17 and reduction of K9 and K14 might contribute to the underlying pathogenesis. Unfortunately, comparison with hand eczema studies is not possible yet, because similar protein expression studies are lacking.

First report of zoonotic Cryptosporidium parvum GP60 subtypes IIaA15G2R1 and IIaA16G3R1 in wild ponies from the northern Iberian Peninsula.

Studies on the prevalence and molecular characterization of Cryptosporidium spp. affecting feral horses are scarce. The highland areas of the northern Iberian Peninsula are home to a large population of wild ponies which generally roam free in the ancient natural range and are subjected to a traditional exploitation regime. In the present study, a total of 79 non-diarrhoeal faecal samples from the wild ponies were collected from the ground immediately after defecation. Cryptosporidium was detected in 10 of the samples (12.6%) by a direct immunofluorescence antibody test and DNA amplification and sequencing. Analysis of partial sequences of the small subunit ribosomal RNA (SSU-rRNA) and heat shock protein (hsp70) loci revealed the presence of Cryptosporidium parvum. In addition, amplification and sequencing of a fragment of the 60-kDa glycoprotein (GP60) locus identified C. parvum subtypes IIaA15G2R1 and IIaA16G3R1. This study reports, for the first time, the occurrence of C. parvum in wild ponies in Europe, specifically in the northern Iberian Peninsula. Identification of the common subtype IIaA15G2R1 and also subtype IIaA16G3R1 (first description) indicates that these hosts may play a role in the sylvatic transmission of C. parvum and that they may act as a reservoir of zoonotic cryptosporidiosis.

Escherichia coli O157:H7 F9 Fimbriae Recognize Plant Xyloglucan and Elicit a Response in Arabidopsis thaliana.

Fresh produce is often a source of enterohaemorrhagic Escherichia coli (EHEC) outbreaks. Fimbriae are extracellular structures involved in cell-to-cell attachment and surface colonisation. F9 (Fml) fimbriae have been shown to be expressed at temperatures lower than 37 °C, implying a function beyond the mammalian host. We demonstrate that F9 fimbriae recognize plant cell wall hemicellulose, specifically galactosylated side chains of xyloglucan, using glycan arrays. E. coli expressing F9 fimbriae had a positive advantage for adherence to spinach hemicellulose extract and tissues, which have galactosylated oligosaccharides as recognized by LM24 and LM25 antibodies. As fimbriae are multimeric structures with a molecular pattern, we investigated whether F9 fimbriae could induce a transcriptional response in model plant Arabidopsis thaliana, compared with flagella and another fimbrial type, E. coli common pilus (ECP), using DNA microarrays. F9 induced the differential expression of 435 genes, including genes involved in the plant defence response. The expression of F9 at environmentally relevant temperatures and its recognition of plant xyloglucan adds to the suite of adhesins EHEC has available to exploit the plant niche.

Cell cycle acceleration and changes in essential nuclear functions induced by simulated microgravity in a synchronized Arabidopsis cell culture.

Zero gravity is an environmental challenge unknown to organisms throughout evolution on Earth. Nevertheless, plants are sensitive to altered gravity, as exemplified by changes in meristematic cell proliferation and growth. We found that synchronized Arabidopsis-cultured cells exposed to simulated microgravity showed a shortened cell cycle, caused by a shorter G2/M phase and a slightly longer G1 phase. The analysis of selected marker genes and proteins by quantitative polymerase chain reaction and flow cytometry in synchronic G1 and G2 subpopulations indicated changes in gene expression of core cell cycle regulators and chromatin-modifying factors, confirming that microgravity induced misregulation of G2/M and G1/S checkpoints and chromatin remodelling. Changes in chromatin-based regulation included higher DNA methylation and lower histone acetylation, increased chromatin condensation, and overall depletion of nuclear transcription. Estimation of ribosome biogenesis rate using nucleolar parameters and selected nucleolar genes and proteins indicated reduced nucleolar activity under simulated microgravity, especially at G2/M. These results expand our knowledge of how meristematic cells are affected by real and simulated microgravity. Counteracting this cellular stress is necessary for plant culture in space exploration.

Evolutionary history and structure of nuclear matrix constituent proteins, the plant analogues of lamins.

Nuclear matrix constituent proteins (NMCPs), the structural components of the plant lamina, are considered to be the analogues of lamins in plants based on numerous structural and functional similarities. Current phylogenetic knowledge suggests that, in contrast to lamins, which are widely distributed in eukaryotes, NMCPs are taxonomically restricted to Streptophyta. At present, most information about NMCPs comes from angiosperms, and virtually no data are available from more ancestral groups. In angiosperms, the NMCP family comprises two phylogenetic groups, NMCP1 and NMCP2, which evolved from the NMCP1 and NMCP2 progenitor genes. Based on sequence conservation and the presence of NMCP-specific domains, we determined the structure and number of NMCP genes present in different Streptophyta clades. We analysed 91 species of embryophytes and report additional NMCP sequences from mosses, liverworts, clubmosses, horsetail, ferns, gymnosperms, and Charophyta algae. Our results confirm an origin of NMCPs in Charophyta (the earliest diverging group of Streptophyta), resolve the number and structure of NMCPs in the different clades, and propose the emergence of additional NMCP homologues by whole-genome duplication events. Immunofluorescence microscopy demonstrated localization of a basal NMCP from the moss Physcomitrella patens at the nuclear envelope, suggesting a functional conservation for basal and more evolved NMCPs.

Immunological Methods for the Detection of Binders in Ancient Tibetan Murals.

Tibetan mural samples from the Jiazhaer mountain cave were studied using enzyme-linked immunosorbent assays (ELISA) and immunofluorescence microscopy (IFM). Samples containing protein binders were first identified using ELISA, and then IFM was used to determine the location of protein binders. Using these methods, we discovered gelatin and casein in samples from wall murals, distributed in both red and black pigments. We excluded the possibility of contamination by conducting further experiments where simulated samples were spiked with milk. We conclude that both gelatin and casein were used as binders in the pigments of the Tibetan Buddhist murals in the Jiazhaer (Transliteration from Tibetan) mountain cave. This is the first evidence of casein being used as a binder in Chinese mural pigments.

Pseudolinear C4d deposits in a hereditary glomerulopathy caused by a rare NC1 collagen-4-alpha-5 missense mutation: a "new disease entity"?

C4d positive glomerulopathies with pseudolinear capillary wall deposits caused by basement membrane (GBM) remodeling have sporadically been reported in renal transplants. Here we describe the case of a hypertensive 60 year-old male with a 5 month history of nephrotic range proteinuria in the setting of normal serum creatinine, complement and ANA levels. Work-up showed MGUS (IgG/kappa restricted). A diagnostic renal biopsy to search for monoclonal gammopathy of renal significance demonstrated thickened glomerular capillary walls with strong pseudolinear complement factor C4d deposits by immunofluorescence microscopy (IF); all other IF studies including stains for Col4A3 were unrevealing with only minor abnormalities seen for Col4A5. The strong and unusual C4d staining of undetermined direct diagnostic significance triggered additional electron microscopic studies uncovering marked structural GBM changes suggestive of a hereditary nephropathy. Further genetic testing revealed a very rare X-linked single missense mutation in the NC1 domain of Col4A5 (exon 51) with a single amino acid substitution (COL4A5 p.A1581S) that has thus far not been reported in hereditary nephropathies. Our case provides further support for pseudolinear glomerular C4d deposits as general markers of GBM remodeling, in our case an unexpected hereditary nephropathy in an older male. Pseudolinear C4d: a general signpost for architectural GBM disturbance and a stimulus for in-depth studies including electron microscopy.

Characterization of a caveolin-1 mutation associated with both pulmonary arterial hypertension and congenital generalized lipodystrophy.

Congenital generalized lipodystrophy (CGL) and pulmonary arterial hypertension (PAH) have recently been associated with mutations in the caveolin-1 ( CAV1 ) gene, which encodes the primary structural protein of caveolae. However, little is currently known about how these CAV1 mutations impact caveolae formation or contribute to the development of disease. Here, we identify a heterozygous F160X CAV1 mutation predicted to generate a C-terminally truncated mutant protein in a patient with both PAH and CGL using whole exome sequencing, and characterize the properties of CAV1 , caveolae-associated proteins and caveolae in skin fibroblasts isolated from the patient. We show that morphologically defined caveolae are present in patient fibroblasts and that they function in mechanoprotection. However, they exhibited several notable defects, including enhanced accessibility of the C-terminus of wild-type CAV1 in caveolae, reduced colocalization of cavin-1 with CAV1 and decreased stability of both 8S and 70S oligomeric CAV1 complexes that are necessary for caveolae formation. These results were verified independently in reconstituted CAV1 -/- mouse embryonic fibroblasts. These findings identify defects in caveolae that may serve as contributing factors to the development of PAH and CGL and broaden our knowledge of CAV1 mutations associated with human disease.